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MicroRNAs (miRNAs) have been implicated in human disorders, from cancers to infectious diseases. Targeting miRNAs or their target genes with small molecules offers opportunities to modulate dysregulated cellular processes linked to diseases. Yet, predicting small molecules associated with miRNAs remains challenging due to the small size of small molecule-miRNA datasets. Herein, we develop a generalized deep learning framework, sChemNET, for predicting small molecules affecting miRNA bioactivity based on chemical structure and sequence information. sChemNET overcomes the limitation of sparse chemical information by an objective function that allows the neural network to learn chemical space from a large body of chemical structures yet unknown to affect miRNAs. We experimentally validated small molecules predicted to act on miR-451 or its targets and tested their role in erythrocyte maturation during zebrafish embryogenesis. We also tested small molecules targeting the miR-181 network and other miRNAs using in-vitro and in-vivo experiments. We demonstrate that our machine-learning framework can predict bioactive small molecules targeting miRNAs or their targets in humans and other mammalian organisms., (© 2024. The Author(s).)

Introduction: Metabolic adaptability, including glucose metabolism, enables cells to survive multiple stressful environments. Glycogen may serve as a critical storage depot to provide a source of glucose during times of metabolic demand during the metastatic cascade; therefore, understanding glycogen metabolism is critical. Our goal was to determine mechanisms driving glycogen accumulation and its role in metastatic (MCF10CA1a) compared to nonmetastatic (MCF10A- ras ) human breast cancer cells., Methodology: 13 C 6 -glucose flux analysis in combination with inhibitors of the gluconeogenic pathway via phosphoenolpyruvate carboxykinase (PCK), the anaplerotic enzyme pyruvate carboxylase (PC), and the rate-limiting enzyme of the pentose phosphate pathway (PPP) glucose 6-phosphate dehydrogenase (G6PD). To determine the requirement of glycogenolysis for migration or survival in extracellular matrix (ECM) detached conditions, siRNA inhibition of glycogenolysis (liver glycogen phosphorylase, PYGL) or glycophagy (lysosomal enzyme α-acid glucosidase, GAA) enzymes was utilized., Results: Metastatic MCF10CA1a cells had 20-fold greater glycogen levels compared to non-metastatic MCF10A- ras cells. Most glucose incorporated into glycogen of the MCF10CA1a cells was in the five 13 C-containing glucose (M+5) instead of the expected M+6 glycogen-derived glucose moiety, which occurs through direct glucose conversion to glycogen. Furthermore, 13 C 6 -glucose in glycogen was quickly reduced (~50%) following removal of 13 C-glucose. Incorporation of 13 C 6 -glucose into the M+5 glucose in the glycogen stores was reduced by inhibition of PCK, with additional contributions from flux through the PPP. Further, inhibition of PC reduced total glycogen content. However, PCK inhibition increased total unlabeled glucose accumulation into glycogen, suggesting an alternative pathway to glycogen accumulation. Inhibition of the rate-limiting steps in glycogenolysis (PYGL) or glycophagy (GAA) demonstrated that both enzymes are necessary to support MCF10CA1a, but not MCF10A- ras , cell migration. GAA inhibition, but not PYGL, reduced viability of MCF10CA1a cells, but not MCF10A- ras , in ECM detached conditions., Conclusion: Our results indicate that increased glycogen accumulation is primarily mediated through the gluconeogenesis pathway and that glycogen utilization is required for both migration and ECM detached survival of metastatic MCF10CA1a cells. These results suggest that glycogen metabolism may play an important role in the progression of breast cancer metastasis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Hicks, Layosa, Andolino, Truffer, Song, Heden, Donkin and Teegarden.)

Background/objectives: Excess adiposity is associated with a higher risk of breast cancer metastasis and mortality. Evidence suggests that dietary vitamin D inhibits breast cancer metastasis. However, the mechanistic link between vitamin D's regulation of adipocyte metabolism and metastasis has not been previously investigated. Therefore, the purpose of these experiments was to examine the effect of the active form of vitamin D, 1α,25-dihydroxyvitamin D (1,25(OH) 2 D), on adipocyte release of bioactive compounds and whether the impact on adipocytes leads to inhibition of breast cancer cell migration, an important step of metastasis., Methods: Differentiated 3T3-L1 adipocytes were treated with 1,25(OH) 2 D for two days, followed by either harvesting the adipocytes or collecting adipocyte-conditioned media without 1,25(OH) 2 D. A transwell migration assay was conducted with vehicle- or 1,25(OH) 2 D-conditioned media. In order to explore the mechanism underlying effects on breast cancer metastatic capability, the mRNA expression of leptin, adiponectin, insulin-like growth factor (IGF-1), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1) was measured in adipocytes following either vehicle or 1,25(OH) 2 D treatment., Results: Conditioned media from 1,25(OH) 2 D-treated adipocytes inhibited the migration of metastatic MDA-MB-231 breast cancer cells compared to conditioned media from vehicle-treated adipocytes. Treatment of adipocytes with 1,25(OH) 2 D decreased mRNA expression of leptin, adiponectin, IGF-1, IL-6, and MCP-1. Consistent with mRNA expression, concentrations of leptin, adiponectin, IGF-1, and IL-6 in adipocyte-conditioned media were decreased with 1,25(OH) 2 D treatment, although MCP-1 remained unchanged., Conclusions: In summary, these results suggest that 1,25(OH) 2 D alters adipocyte secretions to prevent breast cancer metastasis.

Analbuminemia is characterized by the near absence of albumin in the plasma. Different methods are available for measuring albumin levels, but they do not necessarily agree with one another. It is a concern that analbuminemic samples could be falsely characterized due to the incorrect estimation of albumin. The objective of the work was to evaluate the performance of different assays in detecting analbuminemia. Albumin knockout (Alb-/-) mouse plasma was used to test the suitability of different albumin assays for their ability to properly characterize extreme albumin deficiency. Bromocresol green (BCG), bromocresol purple (BCP), enzyme-linked immunosorbent assay (ELISA), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and gel electrophoresis were tested. The LC-MS/MS assay exhibited broad coverage of the amino acid sequence of albumin and indicated 8,400-fold lower (P<0.0001) albumin expression in Alb-/- than wildtype (WT), demonstrating its suitability for identifying extreme albumin deficiency. ELISA estimated albumin at 1.5±0.1 g/dL in WT and was below the detection limit in all Alb-/- samples. Gel electrophoresis yielded consistent results with LC-MS/MS and ELISA. The BCG assay overestimated albumin with apparently appreciable albumin concentrations in Alb-/- mice, yet the assay still indicated a significant difference between genotypes (Alb-/-, 1.2±0.05 g/dL, WT, 3.7±0.1 g/dL, P<0.0001). BCP drastically overestimated albumin and could not successfully identify the known analbuminemic phenotype of Alb-/- mice. By using Alb-/- plasma as a reference material and LC-MS/MS as a reference method, ELISA and gel electrophoresis appear appropriate for identifying analbuminemia, while BCG and BCP are not suitable. It is concluded that dye-binding assays should be avoided when extreme hypoalbuminemia or analbuminemia is suspected., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

An emerging hallmark of cancer is cellular metabolic reprogramming to adapt to varying cellular environments. Throughout the process of metastasis cancer cells gain anchorage independence which confers survival characteristics when detached from the extracellular matrix (ECM). Previous work demonstrates that the bioactive metabolite of vitamin D, 1α,25-dihydroxyvitamin D (1,25[OH] 2 D), suppresses cancer progression, potentially by suppressing the ability of cells to metabolically adapt to varying cellular environments such as ECM detachment. The purpose of the present study was to determine the mechanistic bases of the effects of 1,25(OH) 2 D on cell survival in ECM-detached conditions. Pretreatment of MCF10A-ras breast cancer cells for 3 d with 1,25(OH) 2 D reduced the viability of cells in subsequent detached conditions by 11%. Enrichment of 13 C 5 -glutamine was reduced in glutamate (21%), malate (30%), and aspartate (23%) in detached compared to attached MCF10A-ras cells. Pretreatment with 1,25(OH) 2 D further reduced glutamine flux into downstream metabolites glutamate (5%), malate (6%), and aspartate (10%) compared to detached vehicle treated cells. Compared to attached cells, detachment increased pyruvate carboxylase (PC) mRNA abundance and protein expression by 95% and 190%, respectively. Consistent with these results, 13 C 6 -glucose derived M+3 labelling was shown to preferentially replenish malate and aspartate, but not citrate pools, demonstrating increased PC activity in detached cells. In contrast, 1,25(OH) 2 D pretreatment of detached cells reduced PC mRNA abundance and protein expression by 63% and 56%, respectively, and reduced PC activity as determined by decreased 13 C 6 -glucose derived M+3 labeling in citrate (8%) and aspartate (50%) pools, relative to vehicle-treated detached cells. While depletion of PC with doxycycline-inducible shRNA reduced detached cell viability, PC knockdown in combination with 1,25(OH) 2 D treatment did not additionally affect the viability of detached cells. Further, PC overexpression improved detached cell viability, and inhibited the effect of 1,25(OH) 2 D on detached cell survival, suggesting that 1,25(OH) 2 D mediates its effects in detachment through regulation of PC expression. These results suggest that inhibition of PC by 1,25(OH) 2 D suppresses cancer cell anchorage independence., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Regions of hypoxia are common in solid tumors and drive changes in gene expression that increase risk of cancer metastasis. Tumor cells must respond to the stress of hypoxia by activating genes to modify cell metabolism and antioxidant response to improve survival. The goal of the current study was to determine the effect of hypoxia on cell metabolism and markers of oxidative stress in metastatic (metM-Wnt lung ) compared with nonmetastatic (M-Wnt) murine mammary cancer cell lines. We show that hypoxia induced a greater suppression of glutamine to glutamate conversion in metastatic cells (13% in metastatic cells compared to 7% in nonmetastatic cells). We also show that hypoxia increased expression of genes involved in antioxidant response in metastatic compared to nonmetastatic cells, including glutamate cysteine ligase catalytic and modifier subunits and malic enzyme 1. Interestingly, hypoxia increased the mRNA level of the transaminase glutamic pyruvic transaminase 2 (Gpt2, 7.7-fold) only in metM-Wnt lung cells. The change in Gpt2 expression was accompanied by transcriptional (4.2-fold) and translational (6.5-fold) induction of the integrated stress response effector protein activating transcription factor 4 (ATF4). Genetic depletion ATF4 demonstrated importance of this molecule for survival of hypoxic metastatic cells in detached conditions. These findings indicate that more aggressive, metastatic cancer cells utilize hypoxia for metabolic reprogramming and induction of antioxidant defense, including activation of ATF4, for survival in detached conditions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kiesel, Sheeley, Hicks, Andolino, Donkin, Wendt, Hursting and Teegarden.)

Vitamin D exerts anti-cancer effects in recent clinical trials and preclinical models. The actions of vitamin D are primarily mediated through its hormonal form, 1,25-dihydroxyvitamin D (1,25(OH) 2 D). Previous literature describing in vitro studies has predominantly focused on the anti-tumourigenic effects of the hormone, such as proliferation and apoptosis. However, recent evidence has identified 1,25(OH) 2 D as a regulator of energy metabolism in cancer cells, where requirements for specific energy sources at different stages of progression are dramatically altered. The literature suggests that 1,25(OH) 2 D regulates energy metabolism, including glucose, glutamine and lipid metabolism during cancer progression, as well as oxidative stress protection, as it is closely associated with energy metabolism. Mechanisms involved in energy metabolism regulation are an emerging area in which vitamin D may inhibit multiple stages of cancer progression. LINKED ARTICLES: This article is part of a themed issue on New avenues in cancer prevention and treatment (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.12/issuetoc., (© 2021 The British Pharmacological Society.)

One of the characteristic features of metastatic breast cancer is increased cellular storage of neutral lipid in cytoplasmic lipid droplets (CLDs). CLD accumulation is associated with increased cancer aggressiveness, suggesting CLDs contribute to metastasis. However, how CLDs contribute to metastasis is not clear. CLDs are composed of a neutral lipid core, a phospholipid monolayer, and associated proteins. Proteins that associate with CLDs regulate both cellular and CLD metabolism; however, the proteome of CLDs in metastatic breast cancer and how these proteins may contribute to breast cancer progression is unknown. Therefore, the purpose of this study was to identify the proteome and assess the characteristics of CLDs in the MCF10CA1a human metastatic breast cancer cell line. Utilizing shotgun proteomics, we identified over 1500 proteins involved in a variety of cellular processes in the isolated CLD fraction. Interestingly, unlike other cell lines such as adipocytes or enterocytes, the most enriched protein categories were involved in cellular processes outside of lipid metabolism. For example, cell-cell adhesion was the most enriched category of proteins identified, and many of these proteins have been implicated in breast cancer metastasis. In addition, we characterized CLD size and area in MCF10CA1a cells using transmission electron microscopy. Our results provide a hypothesis-generating list of potential players in breast cancer progression and offers a new perspective on the role of CLDs in cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zembroski, Andolino, Buhman and Teegarden.)

Muscle invasive bladder cancer (MIBC) is a common malignancy and major cause of morbidity worldwide. Over the last decade mortality rates for MIBC have not decreased as compared to other cancers indicating a need for novel strategies. The molecular chaperones HSP70 and HSP90 fold and maintain the 3-dimensional structures of numerous client proteins that signal for cancer cell growth and survival. Inhibition of HSP70 or HSP90 results in client protein degradation and associated oncogenic signaling. Here we targeted HSP70 and HSP90 with small molecule inhibitors that trap or block each chaperone in a low client-affinity "open" conformation. HSP70 inhibitors, VER155008 (VER) and MAL3-101 (MAL), along with HSP90 inhibitor, STA-9090 (STA), were tested alone and in combination for their ability to reduce cell viability and alter protein levels in 4 MIBC cell lines. When combined, VER+MAL synergistically reduced cell viability in each MIBC cell line while not inducing expression of heat shock proteins (HSPs). STA+MAL also synergistically reduced cell viability in each cell line but induced expression of cytoprotective HSPs indicating the merits of targeting HSP70 with VER+MAL. Additionally, we observed that STA induced the expression of the stress-related transcription factor HSF2 while reducing levels of the co-chaperone TTI1., Competing Interests: CONFLICTS OF INTEREST The authors declare no potential conflicts of interest.